Inhibition mechanism of the chloride channel TMEM16A by the pore blocker 1PBC

TMEM16A, a calcium-activated chloride channel involved in multiple cellular processes, is a proposed target for diseases such as hypertension, asthma, and cystic fibrosis. Despite these therapeutic promises, its pharmacology remains poorly understood. Here, we present a cryo-EM structure of TMEM16A in complex with the channel blocker 1PBC and a detailed functional analysis of its inhibition mechanism. A pocket located external to the neck region of the hourglass-shaped pore is responsible for open-channel block by 1PBC and presumably also by its structural analogs. The binding of the blocker stabilizes an open-like conformation of the channel that involves a rearrangement of several pore helices. The expansion of the outer pore enhances blocker sensitivity and enables 1PBC to bind at a site within the transmembrane electric field. Our results define the mechanism of inhibition and gating and will facilitate the design of new, potent TMEM16A modulators.

The solved structure and the proposed mechanism of action (Fig. 6) suggest 1PBC may inhibit ANO1 from the extracellular side, however, the authors exclusively used inside-out patches in their functional tests, in which the drug was applied from intracellular side. How does the drug reach the extracellular binding pocked when it is applied from cytosolic side? It diffuses through the membrane as the authors emphasized that the molecule is non-charged at neutral pH, or it enters the open pore from the intracellular side and reaches the binding pocket in the hourglass-shaped vestibule? How fast the drug can block the current? The important information should be added so that the reader can judge. Also, a non-charge molecule must be membrane permeable (Line 62-63)? Is this true? Please show evidence or cite papers to demonstrate this conclusion. What about the trifluoromethyl group in the molecule?
3. Conclusions not supported by evidence. a. Abstract: Line 12-14: please clarify which evidence showed that "The binding of the blocker shifts the conformational equilibrium towards the open state, revealing a partially open conformation of the channel…". This statement gives an impression that 1PBC (maybe even by itself) facilitates channels entering a partially opens state. However, in the final model (Fig. 6), the authors only depicted that the channels are opened by calcium first and then blocked by 1PBC by binding to its binding site.
b. Line 65-67: "The potency of block increases with more depolarizing voltages (Fig. 1c, d), suggesting that the compound likely acts on the channel from the extracellular side." Why? This was inside-out patch and the drug was applied from intracellular side. How could this data be interpreted to reach the conclusion that 1PBC blocks the channels from extracellular side? c. Line 73-74: "As expected from an open-channel block mechanism, the potency of block increases with an increase in the channel's open probability (Fig. 1e, f)." The proportional increase of blockade with Popen is only one evidence for an open channel blocker. A more standard way of testing is to examine the kinetics of blocking and unblocking in open and closed states. These critical functional tests are missing in the manuscript. d. Line 76-77: "This was incorporated by adding a blocker binding step to the open state in a gating mechanism that we described previously". Please justify the assumption. Why there is only one blocker binding step when the drug is applied from inside whereas it binds to the channel from outside? How valid is this model? f. Line 102-103: "Notably, this conformation now closely resembles the structure of the equivalent region in the paralog TMEM16F." If this is the case, why 1PBC does not block TMEM16F? g. Fig. 3. None of the mutations of the key 1PBC binding resides abolished its inhibitory effects albeit different degrees of shifts of dose responses and alternations of voltage dependence. Would double or triple mutations completely knock out 1PBC sensitivity? It is interesting that all the mutations showed almost identical slopes in the 1PBC dose response curve. Any biophysical meaning?
h. Line 185-186: which evidence supports that G558P stabilizes the closed state of the channel?
i. Line 186-188: "The same mutation … whose conformation was not observed to undergo large rearrangement" . Which structure did the authors refer to? The G558P structure? j. Line 191-192: "…. results from the stabilization of the closed state". Evidence? k. Line 199-200: "In the ca free closed state of the channel, the pore remains constricted throughout and is sterically unfavorable for the access of either anions or the blocker 1PBC…". Did the author have a 1PBC-present closed structure available? If not, this is only a speculation based on the calcium-and 1PBC-free structure.
l. Line 216-217: "… the positive electrostatic environment of the binding site stabilizes the bound inhibitor ….". Observation or speculation? If latter, at least add "may". The same issue applies to the following statements in Line 218-219. m. Line 232-236: " In spite of the dilation of the outer pore, which is now sterically conductive, the narrow pore remains constricted in the 1PBC-bound structure, suggesting that the expansion of the outer pore would precede the widening of the narrow constriction during the transition into a conducting state and that the presented structure might be stabilized in a partially open conformation." What if the blocker enters the binding pocket from the neck region as the inside-out patch recordings demonstrated in the manuscript? Would the statement still hold valid?
a. An essential negative control for the open-channel block model is a calcium free (closed state) EM structure in the presence of 1PBC to show that the drug indeed cannot bind to the protein when not open. Have the authors tried this? Alternatively, does 1PBC binds to the same site in the constitutively activated channel structures (0 Ca) such as I551A (7B5D), which can be blocked by 1PBC in the absence of calcium (Fig. 1g)? These evidence are critical. b. Please explain why a pore blocker like 1PBC (inhibits from both sides) only specifically binds to the pocket in the hourglass-shaped extracellular vestibule? Why not in the neck region if it is an open channel blocker? Did the authors observe other 1PBC binding sites in their EM particles (even though they might be a minor population)?
c. The cartoon representations and color themes in Fig. 3a, 3b, 4c-e, S4 are difficult to read the details. Please improve.

Issues with citations.
A number of studies especially from the An lab have identified the same/similar inhibitor binding pocket in TMEM16A using functional and atomistic simulations. These key references were not even mentioned in this manuscript. 6. Experimental details: a. Electrophysiology: The voltage protocol used in this study were not specified in the legend or methods. Popen is an essential parameter for this study. However, it is unknown how Imax was defined and how the dose response curves were normalized? Although the authors listed different calcium concentrations for different mutations in the supplementary table, it is unclear if these mutations all reached maximum opening at the indicated concentrations. Why not keep it simple by using 100 uM calcium as saturating calcium for all the recordings? b. Fig. 3. Why only K603 was mutated to Gln, while the other residues were mutated to Ala? Please justify.
c. The authors mentioned two mutations that reduce the IC50 without a strong argument for why (line 120). In particular, N546 seems to have the clearest effect and it is not even highlighted in the main text.
d. Line 183-186: Why mutated Gly with Pro? Why not other residues such as Ala? Pro is known to break alpha helces. Pro mutations may introduce unexpected/uninterpretable results. Please justify.
7. Discussions to increase significance. a. Please discuss why TMEM16A has so many different inhibitors based on the authors findings.
b. Please discuss why TMEM16A and TMEM16F have completely different sensitivity to 1PBC. Might be helpful to include a sequence alignment or helical wheels with 16A/B/F in supplementary to more clearly demonstrate the binding pocket differences.
We thank all reviewers for their generally positive and constructive comments, which we have incorporated in our revision and which we have addressed in detail below.

REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author): TMEM16A is an important conductance in human health and disease, and a founding member of the intriguing channel/scramblase TMEM16 superfamily. Unfortunately, TMEM16A pharmacology and an understanding of its structural pharmacology especially has been sorely lacking, largely owing to historical challenges in executing effective ligand discovery against this channel. The elegant study presented here by Lam, Rutz and Dutzler is therefore an important landmark in the field, and no doubt should be considered and prioritized for publication in Nature Communications.

Points to address in revision:
Consider moving some of the intro details and history of 1PBC into the introduction; and also consider comparing it to other known TMEM16A modulators (i.e. are they similar in structure and mechanism, or not).
We have included some introduction of 1PBC in the introduction and added a brief comparison to other known TMEM16A modulators.
Line 54-55: 'Some of these compounds, including 1PBC, consist of aromatic rings, and as weak acids, they are likely to interact with the anion-selective pore.' Comparatively potent inhibitor -this is unusual language. What is the potency? What are you comparing it too? Please remove this language or clarify it.
We have removed the adjective 'comparatively potent'.
How are the authors concluding that 1PBC is neutral at physiological pH? How were the pKa's calculated? Are there QM or other calculations to support these conclusions?
We have mentioned that the pKa's were calculated at the website 'chemicalize.com' in the legend of Fig. 1a and added a reference to the applied method in the main text.
The program empirically predicts the pKa using parameters optimized on a training set of organic molecules with experimentally determined pKa 1,2 . These calculations take into account the effect of partial charges, polarizability, and intramolecular H-bonds.
Line 70-71: '1PBC contains two proton-accepting groups that titrate with acidic and basic pKa's as predicted based on theoretical considerations 52 .' Can the authors clarify what they mean about "indirect mechanisms" (components of) voltage dependent block? And do you expect membrane partitioning effects to be contributing here? Also please clarify how the membrane potential profile is being calculated.
We have clarified that the bulk of the observed voltage dependence of block is due to the binding of the anionic blocker within the electric field and included potential additional sources contributing to the effect.
Line 75-81: 'Since the pore would be too narrow to permit its passage 22 , our results imply that, at neutral pH, the predominantly uncharged 1PBC is freely membrane-permeable, but that it binds to the channel in a deprotonated state within the transmembrane electric field, conferring the bulk of the observed voltage dependence. A closer examination of this voltage dependence reveals a non-monotonic exponential variation of the IC50's ( Fig. 1d), suggesting that additional factors contribute to 1PBC block, potentially originating from interactions with permeating anions or a change in the pore conformation.' We have added in the legends that the membrane potential profile was calculated using the PBEQ module in CHARMM and details of the system can be found in the Methods.
Line 541-542: 'The membrane potential profile was calculated using the PBEQ module in CHARMM (see Methods).' Selectivity of 1PBC -can you please test it against TMEM16B? What is the conservation of residues at the binding site between 16A and 16B? TMEM16F is clearly divergent.
We have included data characterizing 1PBC block for TMEM16B (Fig. 1e and Supplementary Fig. 1a) and provided a sequence alignment in Fig. 1g. 1PBC blocks TMEM16B within the same concentration range with similar voltage dependence and slightly lower potency.
If 1PBC binding is state-dependent (in addition to being voltage-dependent), can the authors comment on why the IC50 is not left shifted with increasing Ca2+ concentration for the WT channel, but there is a left shift of the presumed constitutive activated mutants. I would have anticipated the opposite behaviors.
The IC50 is left-shifted with increasing Ca 2+ concentrations (Fig. 2b) and the amount of shift is in agreement with what is expected for an open-channel block mechanism (Fig. 2b, solid line). The IC50 values were derived empirically from Fig. 2a. This is consistent with a Ca 2+ -dependent change in the affinity of the blocker that is shown in constitutively active mutants (Fig. 2c), demonstrating that a conductive state sampled at zero Ca 2+ has a lower affinity for the blocker.
Congratulations on achieving these improved resolutions and nice maps. Can the authors comment if they noted any change in sequence register in their higher resolution / better ordered TMEM16A structure relative to prior models?
We did not observe any changes in sequence register for other helices except for helix 3, which we have highlighted in Fig. 6  Can the authors please use stronger differences in colors in their figures, e.g. dark green and dark grey for the protein and compound are hard to distinguish (esp. w the heavy use of fading).
We have now used stronger differences in the colors between the different chains and conformations of the protein and a brighter color for the inhibitor (Fig. 3-8).
Does the Ca2+ sensitivity of the studied mutants change? E.g. are the Y514 and Y598 mutants impacted in any way? Should we not expect that the relative Po is changing across these mutants? Please consider the need to address this.
The EC50 of these mutants have been reported in our previous study 3 . The Ca 2+ sensitivity of Y514A is not changed, while that of Y598A is lowered by about 2-fold. We have addressed where appropriate.
Line 191-194: 'Although not having any net energetic effect on activation 27 , the comparatively large impact of truncating the Tyr 514 sidechain on blocker binding (Fig. 5d), which has moved out of the binding site to interact with α4 ( Fig. 6c, d), reflects the importance of this residue in stabilizing the observed channel conformation.'

Position of N546 is not indicated in the figure.
We have now included Asn 546 in Fig. 4.
Can the authors comment on why they believe the 1PBC bound structure has a non-conductive pore. Does this represent a post-open, inactivated or collapsed state of the pore?
In the 1PBC-bound structure, the extracellular vestibule containing the blocker binding site and the adjacent region of the narrow neck have both expanded sufficiently compared to the Ca 2+ -free structure to accommodate Clor even the larger Iwhereas the diameter of the gate region has remained unchanged ( Fig. 6d and Fig. 7a-c). While the neck region in this structure has a dimension that is perhaps sufficient to accommodate a dehydrated Cl -, this region may be too narrow to allow a dehydrated Ito pass through (Fig. 7b, c). For this reason, it is unclear how closely the observed structure would represent a conductive state. Given that the blocker stabilizes a Ca 2+ -dependent rearrangement of the outer vestibule that is also supported by our data on constitutively active mutants ( Fig. 2c and Supplementary Fig. 1c), it is likely that this state is functionally relevant and could represent a pre-open state of the pore that we have previously identified in our kinetic analysis 4 .
From saturating 1PBC concentrations, can the authors please demonstrate the time course for washout of 1PBC at different Ca2+ concentrations? Should we expect that washout / recovery will be slower at lower Ca2+?
We have performed these experiments and observed that the time course of wash out appears to be slower at higher Ca 2+ concentrations ( Supplementary Fig. 2a, b). In addition, the fractional blockade increases with increasing Ca 2+ concentrations, consistent with a higher potency under these conditions ( Supplementary Fig. 2a

The phrase "open-channel block" in the title sounds like an object related to a traffic jam. Perhaps, it should be changed into something like "open-state blockade."
We have modified the title to 'Inhibition mechanism of the chloride channel TMEM16A by the pore blocker 1PBC'.

Is the TMEM16A protein from mouse? I did not find the info in the text.
Yes, our study was performed with the ac splice variant of murine TMEM16A, which we have stated in the 'Methods'.
We have quantified the tilt, it is about 6 degrees.
Line 176-178: 'These differences include an outward movement of the N-terminal part of α4 by about 6° resulting in the displacement of Cα positions of up to 3 Å, leading to a widening of the entrance of the inhibitor binding pocket (Fig. 6c).'

L181, it has previously been observed that glycine works as a hinge for helix bending. The authors may want to cite some published work.
We have added the following sentence and a reference to published work. 5. Figure 2. The density of 1PBC should be shown in a color to make it more distinguished from the protein part. This suggestion also applies to Figure 3.
The density of 1PBC is shown in yellow (Fig. 3). We have changed the color of 1PBC to make it more distinguished from the protein ( Fig. 3-8).

Figure 3, a schematic contact map can be included to show blocker -protein interaction.
We have added a schematic contact map in Fig. 4c.

Figure 4, the moving parts of the helixes can be shown in a more different color.
We have changed the color of the Ca 2+ -free structure ( Fig. 3-8).

ED Table 1. The table needs to include the image collection mode and the energy filter slit value.
In addition, all numbers should be shown in a standard way: 2203806 -> 2,203,806 … We have included the image collection mode and the slit value and have converted the numbers to a standard format (Table 1).

Reviewer #3 (Remarks to the Author):
The Throughout our manuscript, we have exerted caution to ensure that all conclusions are supported by data. This is even strengthened in our revision where we have included novel functional experiments and kinetic modelling. Additionally, we have made an effort to better emphasize the basis of the mechanistic claims in the text.
MAJOR COMMENTS:

Justification.
In the Introduction, the authors mentioned there are many ANO1 modulators and listed several. However, it will be more informative to the readers by including more justifications on why chose 1PBC. This is important. Without a comprehensive justification, the general mechanism that the authors wished to reach cannot be achieved, and "A" needs to be added in front of "Mechanism" in the title.
We have selected 1PBC due to its promising chemical properties that make it an ideal probe for the current study. 1PBC consists of a rigid polycyclic ring system, which eases its identification in the cryo-EM density, and it is among the most potent of the currently described blockers/inhibitors with comparatively high solubility, which permits functional investigations in a broader concentration range.
We have also modified the title to 'Inhibition mechanism of the chloride channel TMEM16A by the pore blocker 1PBC'.

Inconsistency between functional and structural evidence.
The solved structure and the proposed mechanism of action ( Fig. 6) suggest 1PBC may inhibit ANO1 from the extracellular side, however, the authors exclusively used inside-out patches in their functional tests, in which the drug was applied from intracellular side. How does the drug reach the extracellular binding pocked when it is applied from cytosolic side? It diffuses through the membrane as the authors emphasized that the molecule is non-charged at neutral pH, or it enters the open pore from the intracellular side and reaches the binding pocket in the hourglassshaped vestibule? How fast the drug can block the current? The important information should be added so that the reader can judge. Also, a non-charge molecule must be membrane permeable (Line 62-63)? Is this true? Please show evidence or cite papers to demonstrate this conclusion. What about the trifluoromethyl group in the molecule?
The membrane permeability of hydrophobic compounds is a general and widely accepted mechanism in membrane protein pharmacology that permits the access of a compound to a binding site located on the opposite side of its application. Although the solubility of 1PBC is comparatively high in an aqueous environment (~100 µM in aqueous solution) in relation to other TMEM16A blockers, it is non-polar and mostly uncharged and is therefore expected to be membrane permeable. The assumption of being uncharged in solution was based on the described estimation of its pKa values, which are in accordance with the pKa's of heterocyclic nitrogens and phenol groups in common organic molecules.
As shown in our revised manuscript, the kinetics of block is fast (~50 ms at a 1PBC concentration of 3 µM, which is close to its IC50). These data were obtained from ultra-fast perfusion and washout experiments of 1PBC measured in excised patches and are displayed in Supplementary Fig. 2a, b.
The assumption that 1PBC binds to a site located at the extracellular side of the protein in an anionic form is supported by essentially all of our data but also by previous experiments. Our previous analysis of the anion permeation path showed that the pore has a positive electrostatic potential 6,8 and is strongly anion-selective 5,7 and that both properties are determined by basic amino acids bordering the narrow neck region of the pore 7 . These observations support the observed preference of negatively charged compounds to bind to the TMEM16A pore (which is a general property of anion channel blockers). Binding in an anionic form is plausible and is supported by our experimental data. A decrease of the pKa of the hydroxyl in 1PBC is very likely given the general positive electrostatics of the pore and its interaction with the positively charged sidechain of Lys 603. The importance of this interaction is emphasized by the mutation K603Q where the replacement of the positive charge with a polar amino acid strongly decreases the potency of 1PBC and completely abolishes the voltage dependence of block (Fig. 5c). The highly electronegative trifluoromethyl group provides additional stabilization of the deprotonated form of the molecule, which further facilitates the decrease in the pKa.
Beside our structural data, the experimental voltage dependence, which shows an increase of the potency of the blocker at positive potential, provides strong evidence that the blocker binds from the extracellular side. Our results are generally consistent with Peters et al 2015 where 1PBC was applied to the external side of the channel and resulted in the same polarity of voltage dependence 9 , indicating that regardless of which side the compound was applied, it only blocks from the extracellular side. Apart from the observed voltage dependence, which defines the sidedness of block, the diffusion of the blocker across the pore is highly unlikely. Based on our previous data, the narrow neck region is inaccessible to even the small MTS reagent MTSEA 6 , indicating that the more bulky 1PBC is sterically prevented from reaching its binding site from the inside. The fact that 1PBC remains in its binding site in the extracellular vestibule and blocks the channel provides direct evidence that it cannot permeate the channel.
Collectively, the application of the blocker from the inside while acting from the extracellular side thus strongly suggests that it freely diffuses across the membrane.
To make this clearer, we have added the following sentences to our manuscript.
Results, Line 75-78: 'Since the pore would be too narrow to permit its passage 22 , our results imply that, at neutral pH, the predominantly uncharged 1PBC is freely membrane-permeable, but that it binds to the channel in a deprotonated state within the transmembrane electric field, conferring the bulk of the observed voltage dependence.' Discussion, Line 237-238: 'Access from the cytoplasm, in contrast, is impeded by the narrow diameter of the neck, which precludes the diffusion of even smaller solutes 22 .'  Fig. 2c-e). We thus conclude that the blocker preferentially binds the Ca 2+ -bound open state, which is also supported by mutants with basal activity where the potency is much higher in the Ca 2+ -bound state (Fig. 2c). In this respect, it should be emphasized that Fig. 9 (previously Fig. 6) represents a simplified schematic cartoon for the discussion, which illustrates the Ca 2+ dependence of the remodeling of the blocker binding site and the action of the blocker in obstructing the conductive pore after binding to this site. We do not want to imply that the blocker would not bind to a partially open channel where the gate is still closed (i.e. second state from left), which it is probably represented in our structure. We have now explicitly mentioned this in the legend.
Legend of Fig. 9, line 606-607: 'Blocker access to a pre-open conformation, where the site is already remodeled but the gate is still closed, appears to be feasible and might be represented in the observed structure.' b. Line 65-67: "The potency of block increases with more depolarizing voltages (Fig. 1c, d), suggesting that the compound likely acts on the channel from the extracellular side." Why? This was inside-out patch and the drug was applied from intracellular side. How could this data be interpreted to reach the conclusion that 1PBC blocks the channels from extracellular side?
As discussed in detail in point (2), since TMEM16A is an anion channel and the electrostatic potential within its pore is strongly positive, the binding of the blocker presumably occurs in its anionic form.
Depolarization gives rise to a transmembrane electric field that is inside-positive and effectively increases the affinity of the blocker, given that the blocker blocks in its charged form and binds at a site within the transmembrane electric field. The fact that the blocker blocks from the outside even when applied from the inside provides direct experimental evidence that the blocker diffuses across the membrane.
c. Line 73-74: "As expected from an open-channel block mechanism, the potency of block increases with an increase in the channel's open probability (Fig. 1e, f) We have performed these experiments and observed that, while the time course of block depends slightly on Ca 2+ concentrations, the unblocking kinetics is slowed at increasing Ca 2+ concentrations ( Supplementary Fig. 2a, b), indicating an increase in the apparent affinity of the blocker at higher Ca 2+ concentrations. In addition, the fractional blockade increases with increasing Ca 2+ concentrations, consistent with a higher potency under these conditions ( Supplementary Fig. 2a, b). These observations are consistent with a stabilization of the blocked state at higher Ca 2+ concentrations and is in agreement with an open-channel block mechanism (Fig. 2a, b and Supplementary Fig. 2c-e). In contrast, a closedstate antagonism model predicts that increasing Ca 2+ concentrations would antagonize blockade by 1PBC, likely due to the depletion of closed states ( Supplementary Fig. 2f-h). We have included these new results in Supplementary Fig. 2. d. Line 76-77: "This was incorporated by adding a blocker binding step to the open state in a gating mechanism that we described previously". Please justify the assumption. Why there is only one blocker binding step when the drug is applied from inside whereas it binds to the channel from outside? How valid is this model?
The Hill coefficient for 1PBC block is 1 (Fig. 1), which indicates the binding of 1 molecule per pore. A blocking step from the open state is the simplest mechanism that shows an agreement with the steadystate data (Fig. 2a, b), suggesting that such model captures the most essential features of 1PBC block. These are fully supported by the structure where there is one 1PBC molecule bound at each extracellular vestibule (Fig. 3). As discussed in points (2) and (3c), our functional data indicate that the blocker acts from the outside and the fact that it was applied from the inside indicates the membrane permeability of the blocker. As discussed in point (3a), our structural data indicate that blocker binding is accompanied by an expansion of the outer vestibule (Figs. 6, 7), which is consistent with a mutual stabilization of these two events. This agrees well with the IC50 of the blocker at the Ca 2+ concentrations tested, which becomes more potent according to the open probability of the channel (Fig. 2a, b and Supplementary Fig. 2a, b), and is quantitatively consistent with an open-state block mechanism as shown in our modelling results (Fig. 2a, b).
f. Line 102-103: "Notably, this conformation now closely resembles the structure of the equivalent region in the paralog TMEM16F." If this is the case, why 1PBC does not block TMEM16F?
We here only refer to the respective conformations of α3 (up to Arg 515 in TMEM16A), which differ in the previously determined Ca 2+ -bound structures of TMEM16A 6 and F 10 . In our view, the described conformation of TMEM16F does not show an active state since α3 and 4 are both tightly interacting with the remainder of the protein, not forming any cavity that would permit blocker binding 10 . Although we do currently not know the structure of an active conformation of TMEM16F, our results show that the binding of 1PBC is sensitive to the exact conformation of its binding site and our results thus suggest that TMEM16F might not adopt this conformation.
This was discussed in line 280-283.
'Despite the conservation of residues of the extracellular vestibule, 1PBC is selective for TMEM16 channels over the scramblase TMEM16F, a feature that is also reported for the Clchannel inhibitors NFA and NPPB 5 . This is likely a consequence of conformational differences in the region surrounding the binding site, reflecting the distinct functional properties of these paralogs.' We have also clarified in line 120-121: 'Notably, this α3 conformation (up to Arg 515 in TMEM16A) now closely resembles the structure of the equivalent helix in the paralog TMEM16F 31 .' g. Fig. 3

. None of the mutations of the key 1PBC binding resides abolished its inhibitory effects albeit different degrees of shifts of dose responses and alternations of voltage dependence. Would double or triple mutations completely knock out 1PBC sensitivity? It is interesting that all the mutations showed almost identical slopes in the 1PBC dose response curve. Any biophysical meaning?
It is conceivable from the structure that multiple interactions contribute energetically to stabilize the bound blocker, which is also evident from our functional data (Figs. 4, 5). Given the low solubility of 1PBC in aqueous solutions (~100 µM), it would be difficult to establish unambiguously a complete knockout of 1PBC sensitivity. The identical slopes in the dose-response curves, all with a Hill coefficient of 1, indicate that the mutations do not affect the binding stoichiometry of the blocker.

h. Line 185-186: which evidence supports that G558P stabilizes the closed state of the channel?
G558P lowers the Ca 2+ potency by about 3-fold (Fig. 8d). This suggests that the mutation, which is not directly involved in Ca 2+ binding, stabilizes the closed state of the channel, as EC50 is a function of both affinity and efficacy 11 .
i. Line 186-188: "The same mutation … whose conformation was not observed to undergo large rearrangement". Which structure did the authors refer to? The G558P structure?
In this case, the 1PBC/Ca 2+ -bound structure in comparison to the Ca 2+ -free apo structure was referred to. Structural comparison between these two structures suggests that the conformation around Gly 558 does not undergo large rearrangement (Fig. 8a). We have made a reference to Fig. 8a to clarify.
Line 209-211: 'The same mutation did not interfere with block by 1PBC (Fig. 8e), which might be expected for a residue that is remote from the site of inhibition and whose conformation was not observed to undergo large rearrangements (Fig. 8a).' j. Line 191-192: "…. results from the stabilization of the closed state". Evidence?
As discussed in point (3h) and using the same reasoning, a shift in the EC50 in G510P suggests a stabilization of the closed state.

k. Line 199-200: "In the ca free closed state of the channel, the pore remains constricted throughout and is sterically unfavorable for the access of either anions or the blocker 1PBC…". Did the author have a 1PBC-present closed structure available? If not, this is only a speculation based on the calcium-and 1PBC-free structure.
In the Ca 2+ -free apo structure, both the outer pore and the neck region are constricted (Fig. 6e) and the pore diameter is too narrow to accommodate a Clion (Fig. 7c), which is therefore unfavorable for the access of both anions and the blocker 1PBC.
l. Line 216-217: "… the positive electrostatic environment of the binding site stabilizes the bound inhibitor ….". Observation or speculation? If latter, at least add "may". The same issue applies to the following statements in Line 218-219.
Neutralizing either Arg 515 or Lys 603, which are both in direct contact with 1PBC, results in a profound decrease in the potency of 1PBC (Fig. 5c, d). Mutation of the relatively remote Arg 535 also leads to a moderate decrease in the affinity of the blocker (Fig. 5c, d). These data together provide experimental evidence showing that a positive electrostatic environment of the binding site stabilizes the bound blocker.

m. Line 232-236: "In spite of the dilation of the outer pore, which is now sterically conductive, the narrow pore remains constricted in the 1PBC-bound structure, suggesting that the expansion of the outer pore would precede the widening of the narrow constriction during the transition into a conducting state and that the presented structure might be stabilized in a partially open conformation." What if the blocker enters the binding pocket from the neck region as the insideout patch recordings demonstrated in the manuscript? Would the statement still hold valid?
As discussed in points (2) and (3b), the blocker does not access its side from the cytoplasm via the narrow neck but the stabilization of the observed conformation would be independent of the access path.

Questions about structural biology.
a (Fig. 1g)? These evidence are critical.

. An essential negative control for the open-channel block model is a calcium free (closed state) EM structure in the presence of 1PBC to show that the drug indeed cannot bind to the protein when not open. Have the authors tried this? Alternatively, does 1PBC binds to the same site in the constitutively activated channel structures (0 Ca) such as I551A (7B5D), which can be blocked by 1PBC in the absence of calcium
Although 1PBC remains capable of blocking I551A at zero Ca 2+ , the potency is about 10-fold lower than at saturating Ca 2+ , indicating a preference for the Ca 2+ -bound state (Fig. 2c). The smaller voltage dependence at zero Ca 2+ might suggest less penetrance in the transmembrane electric field ( Supplementary Fig. 1c). A similar Ca 2+ dependence, albeit smaller, is observed in the wild-type channel, where 1PBC block is more potent at saturating compared to intermediate Ca 2+ (Fig. 2a, b). This is likely due to a combination of a higher open probability and structural changes around the 1PBC binding site that we observe here. In contrast, a closed-state antagonism model predicts that increasing Ca 2+ concentrations would antagonize blockade by 1PBC ( Supplementary Fig. 2f-h) and is thus not compatible with the functional data.
We respectfully disagree that a structure of a Ca 2+ -free state in presence of the blocker would provide an essential negative control. The conclusive localization of small molecules in cryo-EM data, as shown here, is still a large experimental challenge and far from routine. In this respect, we want to emphasize the enormous effort behind the structure determination of the 1PBC-bound conformation, which required extensive data collection from samples applied to grids with different chemical properties to be able to come up with a model of the complex that allows the localization of the inhibitor, which does not necessarily work in every case. Whereas our data have unambiguously revealed the conformation of TMEM16A in an inhibited state, the mere absence of density does not itself provide any strong evidence. Since the goal of the present study is to understand the main mechanism of 1PBC block, with structural and functional data being in strong accordance, a Ca 2+ -free structure in the presence of the inhibitor would be inconclusive and would not change the interpretation of our data and is thus beyond the scope of our current study.

b. Please explain why a pore blocker like 1PBC (inhibits from both sides) only specifically binds to the pocket in the hourglass-shaped extracellular vestibule? Why not in the neck region if it is an open channel blocker? Did the authors observe other 1PBC binding sites in their EM particles (even though they might be a minor population)?
As discussed in points (2), (3b), and (3d), there is only one binding site (Hill coefficient is 1) that is accessible only from the outside (block is facilitated by depolarization) and 1PBC has a dimension that does not allow it to enter the narrow neck region (hence functioning as a channel blocker). The observation that a single 1PBC molecule binds to the extracellular vestibule is therefore consistent with its functional properties. We did not observe other 1PBC binding sites and there is no evidence that 1PBC would block the channel from the cytoplasm. Fig. 3a, 3b, 4c-e, S4 are difficult to read the details. Please improve.

c. The cartoon representations and color themes in
We have modified the mentioned figures and changed the colors of the models to make them easier to distinguish (Fig. 3-8).

Issues with citations.
A number of studies especially from the An lab have identified the same/similar inhibitor binding pocket in TMEM16A using functional and atomistic simulations. These key references were not even mentioned in this manuscript.
The referred studies have used a combination of docking and molecular dynamics simulations to localize putative inhibitor binding sites that are extracellular to the site observed in this study. In the deposited structures of TMEM16A, which served as the basis for these studies, this region is mobile and poorly defined in the cryo-EM density. Additionally, the 1PBC binding site described here is not available in these structures since α3 was modeled in a 'down' conformation where the binding site is blocked by Tyr 514. Nevertheless, it is remarkable that the work has identified a related region for inhibitor interactions.
We have thus added two citations and made the following changes to our manuscript: We have added the voltage protocol in Fig. 1b. The I-V curves in the presence of increasing concentrations of 1PBC were calculated as a fraction of the I-V curve obtained in the absence of the blocker that was obtained within 5 seconds prior to the application of the blocker, which results in the concentration-response curves. We can confirm that all 1PBC experiments were performed at saturating Ca 2+ as we have determined their EC50 in our previous study 3 and as the currents do not show voltagedependent relaxation at the indicated Ca 2+ , a hallmark of saturating Ca 2+ . Using 100 µM Ca 2+ will result in unnecessarily fast current rundown, which will adversely affect the measurements.
We have added in line 401-402: 'Concentration-response relations, obtained from the ratio of the I-V plots before and after the application of the blocker, were fitted to the Hill equation…' b. Fig. 3. Why only K603 was mutated to Gln, while the other residues were mutated to Ala? Please justify.
The currents of K603A were very low and the mutant was thus not suitable to characterize 1PBC block. The current size of the mutant K603Q, in contrast, was higher, though it was still challenging to measure 1PBC block. We therefore characterized K603Q to investigate the effect of neutralizing the positive charge of Lys 603.
c. The authors mentioned two mutations that reduce the IC50 without a strong argument for why (line 120). In particular, N546 seems to have the clearest effect and it is not even highlighted in the main text.
We have highlighted in line 139-142 the polar properties of the two residues Gln 637 and Asn 546, which are presumably a reason for the increase in the potency of 1PBC given that the blocker is stabilized by the surrounding hydrophobic residues (Fig. 5).
Line 139-142: 'In contrast, the surrounding non-charged polar residues (i.e. Thr 539, Asn 546, and Gln 637) have less or even an opposite energetic contribution, except for Thr 539, which engages in an interaction with the trifluoromethyl group of 1PBC (Fig. 5a-d).'

d. Line 183-186: Why mutated Gly with Pro? Why not other residues such as Ala? Pro is known to break alpha helces. Pro mutations may introduce unexpected/uninterpretable results. Please justify.
We mutated the glycines to prolines as an attempt to strongly reduce backbone flexibility. The same approach was also used in our previous study to investigate the role of Gly 644 on channel activation 6 .
Although prolines cannot form the canonical backbone interactions and thus often introduce kinks in αhelices, they are frequently found in regions with α-helical properties (e.g. Pro 658 in helix 6).
This was justified in line 206-207: 'Replacing Gly 558 with the more rigid proline exerts appreciable effects…'

Discussions to increase significance.
a. Please discuss why TMEM16A has so many different inhibitors based on the authors findings.
We have discussed on the possibility of reported inhibitors in light of our structure, although we refrain from speculating too much given that the functional mechanisms of many of the reported compounds are not very well characterized.
We have added in line 240-242: 'Given the specific interactions between the channel and 1PBC, different mechanisms might be needed to account for the reported inhibition of TMEM16A by structurally unrelated compounds.' b. Please discuss why TMEM16A and TMEM16F have completely different sensitivity to 1PBC. Might be helpful to include a sequence alignment or helical wheels with 16A/B/F in supplementary to more clearly demonstrate the binding pocket differences.
We have discussed the potential conformational difference of the TMEM16A and F (line 280-285) and have included a sequence alignment in Fig. 1g.
Line 280-285: 'Despite the conservation of residues of the extracellular vestibule, 1PBC is selective for anion channels of the TMEM16 family over the scramblase TMEM16F, a feature that is also reported for the Clinhibitors NFA and NPPB 5 . This is likely a consequence of conformational differences in the region surrounding the binding site, reflecting the distinct functional properties of these paralogs.' c. Please also comment on numerous reports that showed that the same inhibitors such as niclosamide, CaCCinh-01, T16Ainh -A01 can suppress both TMEM16A and TMEM16F current.
To our knowledge, there has not been extensive evidence suggesting that these compounds would inhibit both TMEM16A and F current. In fact, in line with our finding with 1PBC, the common chloride channel inhibitors NPPB and NFA have been shown to selectively inhibit TMEM16A but not F 12 . In light of these observations, we have now commented in line 280-283: 'Despite the conservation of residues of the extracellular vestibule, 1PBC is selective for anion channels of the TMEM16 family over the scramblase TMEM16F, a feature that is also reported for the Clchannel inhibitors NFA and NPPB 5 .'

MINOR/OPTIONAL COMMENTS:
1. Abstract: Line 12: please specify the meaning of "chemically similar compounds"?
We have modified the phrase to 'structural analogs' in line 15: 'A pocket located external to the neck region of the hourglass-shaped pore is responsible for openchannel block by 1PBC and presumably also by its structural analogs.' 2. Line 45: more commonly used ANO1 inhibitors such as CaCCinh-01, T16Ainh -A01, and Niclosamide, were not included and cited.
We have now clarified in line 55-58: 'However, the location of their binding sites and the conformations of the channel to which these compounds bind are not known, limiting our ability to design more potent and specific drugs that target TMEM16 proteins.' Fig. 1 is strange. Please rearrange.

The order of the panels in
We have rearranged.
6. Line 64-65: "1PBC blocks TMEM16A completely with an IC50 of ~4 μM at zero mV at physiological salt concentrations" As calcium is a variable, please specify concentration here.
We have changed in line 71-73: 'When applied from the intracellular side, 1PBC blocks TMEM16A completely with an IC50 of ~4 µM at zero mV at a saturating Ca 2+ concentration (2 µM) (Fig. 1b, c).' 7. Line 67-69: "A closer examination of the voltage dependence reveals a non-monotonic exponential variation of the IC50's (Fig. 1d), suggesting that 1PBC block might consist of different sources of voltage dependences including those that are conferred via indirect mechanisms." Please explain what direct and indirect mechanisms are, and postulate why it follows a nonmonotonic exponential.
Direct indicates an intrinsic voltage dependence of the blocker due to the binding site being located within the transmembrane electric field, and indirect refers to voltage dependence for reasons not related directly to the location of the binding site.
We have included potential mechanisms in line 78-81: 'A closer examination of this voltage dependence reveals a non-monotonic exponential variation of the IC50's (Fig. 1d), suggesting that additional factors contribute to 1PBC block, potentially originating from interactions with permeating anions or a change in the pore conformation.' 8. Line 69-70: "This inhibitor appears to be selective for TMEM16 channels, as it is ineffective in blocking the current mediated by the scramblase TMEM16F …". This description is not accurate. First, "selective" for which TMEM16 channels? Second, the authors only tested 1PBC's effect on TMEM16F current, not its lipid scrambling.
We have now included experiments with TMEM16B, which is also blocked by 1PBC at similar concentrations ( Fig. 1e and Supplementary Fig. 1a). We have only tested the effect of 1PBC on TMEM16F in electrophysiological experiments, therefore we made the description that it does not block the current mediated by TMEM16F and have made no reference to lipid scrambling.
9. Line 78: please specify the calcium concentration used (should be 2 uM) and justify why chose this concentration.
We have specified the Ca 2+ concentration in line 73 '(2 µM)' and justified it as a saturating concentration.
'When applied from the intracellular side, 1PBC blocks TMEM16A completely with an IC50 of ~4 µM at zero mV at a saturating Ca 2+ concentration (2 µM) (Fig. 1b, c).' 10. Line 80-81: "The quantitative agreement between the model and the data confirms that the Ca2+ dependence of block is due to a difference in open probabilities." Which one was referred to as "model" and which one 'data'. Which figure did the authors refer to?
We have made a reference to Fig. 2a and b for this statement. The fitted model is shown as solid lines and data are shown as symbols, which we have fully described in the legends to the panels (Fig. 2a, b). 'The agreement between the model and the data confirms that the Ca 2+ dependence of block is due to a difference in open probabilities (Fig. 2a, b and Supplementary Fig. 2c-e).' 11. Fig.1 g and h, why the author choose 0 mV instead of 80 mV, which was used in other panels of Fig.1? Please show WT dose response curves as a reference.
The voltage dependence of 1PBC is different amongst these mutants ( Supplementary Fig. 1c), hence we plotted the data at zero voltage to compare the effect of 1PBC in the absence of voltage (Fig. 2c). We have shown the WT concentration-response relations, which might be obscured by the curves in the presence of Ca 2+ because they are not changed compared to the WT (Fig. 2c).

Line 104:
The authors should remove the statement in line 104 "non-protein density, which is not present in any previous maps of TMEM16A" given that the overall resolution for the other structures are around 4A.
The overall resolution of several other structures (5OYB, 7B5C, and 7B5D) are 3.75, 3.7, and 3.3 Å respectively 3,6 . This density is already evident in our present structure in an earlier reconstruction at ~3.5 Å and is therefore not due to a difference in resolution. Hence, this statement is valid.
13. Line 115: " …and potentially also influences the protonation state of titratable groups of the blocker." If this is a speculation, please move it to discussion section.
It is well understood that the protonation state of a titratable group is under the influence of the electrostatic potential of its surrounding environment 13 .
Mutation to alanine is effectively truncating the functional group of a sidechain.
15. Line 172: Val 508, Val 511, and Ile 512 were mentioned, but no figure or data showed these mutations.
We have added data for Val 511 and Ile 512 in Fig. 5c, d. Like the neighboring aliphatic residues, these mutations also substantially lower the potency of 1PBC (Fig. 5).

Line 243: shifts.
We have changed accordingly.
Line 277-280: 'The binding of Ca 2+ and the blocker shifts the conformational equilibrium towards the open state in a process that involves the movement of several pore helices, which, although pronounced, are less extensive than observed in fungal family members functioning as lipid scramblases 55,56 .'

Peters et al (PNAS, 2015) showed that another two basic residues R621 and R788 enhances the binding affinity of 1PBC. Please discuss what could be the mechanism?
Since these residues are far from the 1PBC binding site, it is not immediately clear how they can affect the binding affinity of 1PBC.
2) Please add some description to explain the gray, orange and blue dots in the cartoon in panel C. Please also draw a cartoon in f to help the readers to understand the model.  1996). In addition, MTSEA or ET are positively charged. It will be unfavorable for these compounds to enter the chloride channel pore. Therefore, this argue seems not be very helpful to exclude the possibility that 1PBC cannot go through and block at the more extracellular portion of the pore.
7) The above comments do not mean the reviewer is not convinced that 1PBC binds to the identified extracellular site. Just hope the authors be aware there might be alternative mechanisms that the current set of experimental evidence has not completely ruled out.
Our response to the final remarks by reviewer #3 is provided below. While we have introduced most suggested modifications, we disagree with the statement in points 6 and 7 and summarize all current experimental evidence supporting our claim that the open pore of TMEM16A would be too narrow to permit diffusion of 1PBC. In fact, it is a common property of most pore blockers to exceed the size of the narrowest section of the pore, which renders them impermeable. These blockers commonly bind outside of the pore constriction in the narrowing funnel where they occlude the entrance to the pore to prevent ion conduction as found in case of 1PBC.

Reviewer #3 (Remarks to the Author):
This reviewer appreciate the authors took tremendous efforts to revise the manuscript. Several additional comments are listed below. Once the authors address these minor concerns, this work will be another milestone in the field. 2. Line 83, please add "CaCC" after "TMEM16" to be more accurate. Other TMEM16 proteins with scramblase activities are also channels.
We have added in line 82 '1PBC appears to be selective for anion channels of the TMEM16 family…'

Line 268 effects -> affects
We have changed in line 266 'The ability of α3 to alter its conformation during gating, which on its extracellular side affects the pore geometry…'

Fig. S2. Please add experimental details and clarification to help the readers to interpret the new results.
We have added more experimental details in the legend of Supplementary Fig. 2 The holding voltage was +80mV. The Po difference between these two Ca 2+ concentrations is about 1.5-2 fold at this voltage. Note that all displayed normalized currents are positive and that they decrease upon perfusion of 1PBC.
We have added to Supplementary Fig. 2  2) Please add some description to explain the gray, orange and blue dots in the cartoon in panel C. Please also draw a cartoon in f to help the readers to understand the model.
We have added an illustration for panel f and made descriptions on the graphical features shown in the illustrations in panels c and f.

3) How was the tau calculated?
The absolute current under each calcium or normalized to the steady state current? Please specify.
The tau values were estimated by an empirical fit consisting of a sum of two exponentials as described in the legend. The current traces were normalized to the magnitude prior to blocker application, which is denoted as I/I0.
We have changed in Supplementary Fig. 2 legend, line 24-26 'The current traces were corrected for rundown using a linearly decaying baseline, and were normalized to the respective steady-state currents in the absence of 1PBC (I/I0).' We have changed in Supplementary Fig. 2 legend, line 33-35 'Time constants of blocking and unblocking and fractional inhibition empirically determined from the calculated time course (d) via a fit to a sum of two exponentials.' 4) Even if 1PBC, as the authors explained in the manuscript, is membrane permeable, seeing almost instantaneous inhibition (ms) is still surprising to this reviewer, given that the molecule needs to penetrate the bilayer, maybe diffuse into the bulk pipette solution and then eventually binds to the extracellular binding site. It seems the authors did not count this potential delay in their kinetic model especially on tau-on in their modeling (panels c-e)?
The diffusion of the blocker across the membrane appears to be fast. Although it is possible that diffusion has an effect on tauon, the incorporation of a time course for the concentration rise of the blocker would not affect the fundamental features of these models. The Kd of ~3.6 µM was estimated from our data (Fig. 2a, b). We used the same Kd for the closed state antagonism model to allow a comparison between the two scenarios. While the Kd determines the 1PBC concentration dependence, it does not affect the opposite trends predicted using these models.

5) Panel f predicts a Closed
We have changed in Supplementary Fig. 2 legend, line 38-39 '…the values of the blocking parameters were: Kd of 1PBC = 3.6 µM (as determined in Fig.  2a, b) and kon = 1 x 10 6 M -1 s -1 . The same values were used for the two models to allow a direct comparison.'

6)
There is no open TMEM16A structure available. The exact dimension of a fully open channel is unknown. The possibility of 1PBC going through an open pore to reach the extracellular binding site cannot be entirely excluded. The authors argued in the rebuttal letter that "Based on our previous data, the narrow neck region is inaccessible to even the small MTS reagent MTSEA6, indicating that the more bulky 1PBC is sterically prevented from reaching its binding site from the inside.". However, MTSEA is well-known to be membrane permeable  . 797-804, 1996). In addition, MTSEA or ET are positively charged. It will be unfavorable for these compounds to enter the chloride channel pore. Therefore, this argue seems not be very helpful to exclude the possibility that 1PBC cannot go through and block at the more extracellular portion of the pore.
Although at the current stage the detailed structure of a fully active state of TMEM16A is presumably unknown, there is ample evidence from structural and functional experiments (including results obtained in the present study and a previous characterization of an activating mutant) that the known Ca 2+ -occupied structures are close to an activated state and probably only require a moderate expansion of the neck to become conductive.
Previous functional studies have provided following evidence for a narrow constriction in the open TMEM16A pore.
1) MTSEA modified the mutant K588C at the inner pore entrance at the boundary between the intracellular vestibule and the narrow neck but not the double mutant K588Q/S592C that is located just one helix turn further into the neck, indicating the inaccessibility of S592 under the same electrostatic conditions even if MTSEA is positively charged.
2) The same conclusion could be drawn using the negatively charged MTSES, which did not modify S592C.
3) Methanesulfonate, which has a valence of -1 and a longest dimension of ~3.5 Å, shows negligible permeability through TMEM16A in ion substitution experiments, indicating that even such a small anion does not permeate the channel.
4) The observation that 1PBC blocks the channel even at strongly positive voltage indicates substantial steric hindrance. Hence the pore diameter is likely considerably smaller than the dimension of 1PBC.
5) The observed voltage dependence indicates that the negatively charged blocker binds from the outside (an opposite polarity would be observed for a block from the intracellular side). In the unrealistic case where the blocker would diffuse through the channel, blockade with the observed properties would not be realized because blocker dissociation would be promoted by both depolarizing and hyperpolarizing voltages.
6) The electrical distance of 1PBC block in functional experiments is 0.2-0.25, which agrees very well with the fractional transmembrane electric potential of 0.2-0.25 at the 1PBC binding site from the outside calculated using the experimental structure (shown in Fig. 3e). Together, these observations strongly suggest that our data are best described by a mechanism where the blocker binds from the extracellular side to a site located within the transmembrane electric field and directly blocks the pore by virtue of its molecular dimension that is incompatible with permeation.

7)
The above comments do not mean the reviewer is not convinced that 1PBC binds to the identified extracellular site. Just hope the authors be aware there might be alternative mechanisms that the current set of experimental evidence has not completely ruled out.
We slightly modified our text (line 75-78) 'Since the pore would most likely be too narrow to permit its passage 22 , our results imply that, at neutral pH, the predominantly uncharged 1PBC is freely membrane-permeable, but that it binds to the channel in a deprotonated state within the transmembrane electric field, conferring the bulk of the observed voltage dependence.'